This application is related to International Patent Application No. PCT/US92/07111, filed on Aug. 24, 1992, which describes materials and methods useful in the prevention and treatment of Human Immunodeficiency Virus (HIV-1) infection and, specifically, monoclonal antibodies useful in passive immunisation of HIV-1 susceptible or infected animals, especially humans. This application gives a background to HIV-1 infection and its prophylactic or therapeutic treatment by passive immunisation, the main points of which are reiterated here.
The infective process of HIV-1 in vivo has been reviewed by McCune Cell, 64 pp. 351-363 (1991). HIV-1 infects cell lineages which express the CD4 receptor. Most of these cells are quiescent, only dividing in response to specific signals, and so HIV-1 infection may cause CD4.sup.+ cells to replicate whereupon viral particles are produced, spreading the infection. Since it is inadvisable to stimulate the immune response of an HIV-1 infected animal, it may be that the best way to prevent or treat HIV-1 infection is by passive immunisation. This involves the administration of anti-HIV-1 antibody to the patient, and presumably, it would be desirable for this antibody agent to be non-immunogenic. There is a precedent for such treatment. Human patients suffering advanced acquired immunodeficiency syndrome (AIDS), the syndrome of progressive immune system deterioration associated with HIV-1 infection, were given plasma containing antibodies against HIV-1 and showed a temporary reduction in various disease parameters (Jackson et al., Lancet, 2, pp. 647-652, 1988). Also the administration of HIV-1 specific antibody to a chimpanzee, prior to exposure to HIV-1, resulted in the animal remaining free of signs of viral infections (Emini et al., Nature, 355, pp. 728-730, 1992).
The HIV-1 major external envelope glycoprotein, gp120, binds to cellular CD4 receptor and facilitates the internalisation of the virus. Several epitopes of gp120 have been associated with the development of neutralizing antibodies with the so-called "principle neutralizing determinant" being localised to the "V3 loop" of gp120, as referenced in the previous Application (PCT/US92/07111). The V3 loop consists of a hypervariable domain which is established by disulphide bonding between cysteine residues flanking the domain.
The PCT Published Patent Application No. PCT/US92/07111 cites examples of antibody reactive to V3 loop which are isolate or type specific and also candidates as broadly neutralizing antibodies, although none of these were proven to neutralize multiple strains of live HIV-1. The patterns of reactivity shown by these antibodies are likely to be related to the array of primary sequences and conformations of the V3 loop. The aforementioned Application also highlights potential inadequacies of such antibodies: CD4 may not be the only cellular receptor responsible for viral infectivity (Cheng-Mayer et al., Proc. Natl. Acad. Sci. U.S.A., 84, pp. 3526-3530, 1987) and antibody/HIV-1 complexes may enhance virus replication by, for example, promoting the infection of monocytes via receptor-mediated endocytosis (Takeda et al., Science, 242, pp. 580-583, 1988).
Previous work had shown that certain animal viruses are inactivated by complement, particularly Clq, through an antibody-independent mechanism (see, for example Weiss, in Molecular Biology of Tumour Viruses, RNA Tumour Viruses, Weiss et al., Eds., Cold Spring harbour Laboratory, New York, pp. 1219-1220, 1982). While Banapour et al., Virology, 252 pp. 268-271 (1986) describe unheated serum preparation as having no effect on the density of HIV-1 or its ability to infect peripheral blood mononuclear cells, Spear et al., J. Virol., 64, pp. 5869-5873 (1990) report that HIV-1 treated with a combination of complement and pooled sera from HIV-1 sero-positive patients exhibits reduced infectivity.
Thus the background to the previous invention (PCT/US92/07111) was the identification of a need for new monoclonal antibody substances which are specifically immunoreactive with HIV-1 and which, preferably, would neutralize multiple HIV-strains. As candidates for use in passive immunisation of infected and non-infected patients, these agents would ideally be able to mediate complement-dependent virolysis of HIV-1 particles and antibody-dependent cytolysis of HIV-1 infected cells.
The aforementioned invention (PCT/US92/07111) provides monoclonal antibodies reactive to the portion of HIV-1 gp120, or the precursor gp160, comprising the amino acid sequence GPGR, characterised by their capacity to neutralize the infection of H9 cells in culture by live HIV-1 strains MN and III.sub.B and to mediate complement-dependent virolysis of HIV-1 particles and/or antibody-dependent cellular cytotoxicity of HIV-1 infected cells. It was suggested that the monoclonal antibodies of the invention would be suitable for use in anti-HIV-1 treatment of animals, especially humans, susceptible to or infected with HIV-1. Within the contemplation of the invention was the similar use of chimeric antibodies, humanized antibodies, antibody fragments or bispecific antibodies, which could be made as derivatives of the antibodies of the invention, and the use of products of the invention in combination with other immunological and/or therapeutic agents.
The invention describes generation of monoclonal antibodies by immunization of a suitable host with live HIV-1, thus presenting gp120 in its native conformation. It is illustrated by the murine monoclonal antibody, NM-01, produced by the hybridoma cell line HB 10726. The efficacy of NM-01 in in vitro assays, the mapping of the epitope of NM-01 to the sequence GPGR of the gp120 V3 loop and the determination of the NM-01 heavy and light chain variable region sequences are described.
It might be expected that a human antibody would be more suitable than a xenogeneic antibody in prophylactic and therapeutic treatment of HIV-1 infection in humans. This is because the human antibody would be less immunogenic than, for example, a murine counterpart (Bruggermann et al., J Exp Med, 170, pp. 2153-2157, 1989) and may, depending on the isotype involved, be more efficient at triggering complement-dependent virolysis and antibody-dependent cellular cytotoxicity of HIV-1 infected cells (discussed by Winter and Milstein Nature, 349, 293-299, 1991).
To secure the advantages of a human antibody, whilst making use of the antigen-binding properties of an antibody raised in a different species, workers have used the technique of humanization to transfer the antigen-binding loops to a human template (for example Riechmann et al., Nature, 332, pp. 323-327, 1988; Tempest et al., Bio/Technology, 9, pp. 266-271, 1991). These loops, known as complementarity determining regions (CDRs) are mounted on a scaffold--the frameworks regions--which together make up the so-called variable domains, situated at the N-terminal ends of each antibody chain. Each binding site is formed, in the most part, from three heavy chain and three light chain CDRs, although framework residues can interact with antigen, either directly or indirectly, by altering the CDR conformation. Genes encoding these recombinant antibodies are expressed in, for example, mammalian cells, and their constant region components can be tailored to suit the application.